Itaconic Acid Production Research Articles

Biomass Demineralization and Pretreatment Strategies to Reduce Inhibitor Concentrations in Itaconic Acid Fermentation by Aspergillus terreus

Itaconic acid (IA) is a platform chemical, derived from non-petroleum sources, produced through the fermentation of glucose by Aspergillus terreus. However, producing IA from alternative sugar sources (e.g., lignocellulose) has been shown to be problematic, requiring post-hydrolysis mitigation to allow growth and IA production by the fungus. It is well known that the side products of lignocellulosic biomass conversion to sugars act as microbial growth inhibitors. An uncommon feature of fungal organic acid fermentations is production inhibition caused by mineral ions in biomass hydrolysate after pretreatment and enzymatic hydrolysis. To minimize mineral introduction during pretreatment and hydrolysis, we determined the sources of growth and production inhibitors at each of these steps. Biomass demineralization and four pretreatment strategies were evaluated for inhibitor introduction. Dilution assays determined the approximate degree of inhibition for each hydrolysate. An ammonium hydroxide pretreatment of demineralized wheat straw presented the lowest concentration of inhibitors and concomitant lowest inhibition: subsequent fermentations produced 35 g L−1 IA from wheat straw hydrolysate (91 g L−1 sugar) without post-hydrolysis mitigation.

Bioconversion of mango peels into itaconic acid through submerged fermentation and statistical optimization of parameters through response surface methodology.

Itaconic acid is an industrially crucial organic acid due to its broad range of applications. The main hurdle in itaconic acid production is the high cost of the substrate, i.e., pure glucose, required for the fermentation process. Pakistan annually produces about 1.7 to 1.8 million metric tonnes of mango fruit. Keeping this in view, the potential of a sugar-rich fruit by-product, i.e., mango peels, was analyzed to be used as a substrate for the biosynthesis of itaconic acid using Aspergillus niger by submerged fermentation. Different physicochemical parameters (incubation period, temperature, agitation rate, inoculum size, and pH) were optimized using the central composite design (CCD) design of response surface methodology (RSM). The maximum production of itaconic acid, i.e., 4.6 g/L, was analyzed using 10% mango peels w/v (water hydrolysate), 3 mL inoculum volume after 5 days of fermentation period at pH 3, and a temperature of 32 °C when the media was kept at a 200-rpm agitation speed. The itaconic acid extraction from mango peels was done using the solvent extraction method using n-butanol. The identification and quantification of itaconic acid produced in the study were done using the Fourier Transform Infrared Spectroscopy (FTIR) spectrum and the High-Performance Liquid Chromatography (HPLC) method. According to HPLC analysis, 98.74% purity of itaconic acid was obtained in the research. Hence, it is concluded from the results that sugar-rich mango peels can act as a promising substrate for the biosynthesis of itaconic acid. Further conditions can be optimized at the bioreactor level to meet industrial requirements.

Understanding the metabolic nodes in itaconic acid synthesis of an Aspergillus terreus mutant identified by high-throughput screening

Aspergillus terreus is the most important strain for the industrial production of itaconic acid. Few works have reported the critical metabolic nodes in the synthesis of itaconic acid in A. terreus. Herein a high-throughput screening method was established to screen an efficient mutant A. terreus 6–12#, which could produce 28.6 g/L itaconic acid with a productivity of 0.36 g/g glucose, about 66 % and 57 % higher than that of the parent strain. On this basis, the expression of the key enzymes was analyzed centering on the glycolytic pathway, the precursor synthesis pathway, the itaconic acid synthesis and degradation pathways, and the membrane transport system. It was proven that the expressions of enzymes favoring itaconic acid synthesis in each of these modules were significantly elevated in the mutant. But the enzymes CAD, MTT, MFS in the itaconic acid synthesis pathway and the membrane transport system displayed low elevation levels, while the degradation pathway was activated with the improvement of itaconic acid, which was considered the possible limited step in itaconic acid synthesis in A. terreus. These findings provided potential metabolic modification targets for further acquisition of excellent A. terreus cell factories and helped to facilitate the industrial production of itaconic acid.

Tumor-associated macrophage-derived itaconic acid contributes to nasopharyngeal carcinoma progression by promoting immune escape via TET2

Nasopharyngeal carcinoma (NPC) is a malignant tumor of epithelial origin in head and neck with high incidence rate in South China, Southeast Asia and North Africa. The intervention of tumor-associated macrophages (Mφs) (TAMs)-mediated immunosuppression is a potential therapeutic strategy against tumor metastasis, but the exact mechanisms of TAM-mediated immunosuppression in nasopharyngeal carcinoma are unclear. Furthermore, how TAM affects the occurrence and development of nasopharyngeal carcinoma through metabolism is rarely involved. In this work, we revealed that NPC cells promoted M2-type Mφ polarization and elevated itaconic acid (ITA) release. Also, TAMs facilitated NPC cell proliferation, migration, and invasion through immune response gene 1 (IRG1)-catalyzed ITA production. Then, IRG1-mediated ITA production in TAMs repressed the killing of CD8+ T cells, induced M2-type polarization of TAMs, and reduced the phagocytosis of TAMs. Moreover, we demonstrated ITA played a tumor immunosuppressive role by binding and dampening ten-eleven translocation-2 (TET2) expression. Finally, we proved that ITA promotes NPC growth by facilitating immune escape in CD34+ hematopoietic stem cell humanized mice. In Conclusion, TAM-derived ITA facilitated NPC progression by enhancing immune escape through targeting TET2, highlighting that interfering with the metabolic pathway of ITA may be a potential strategy for NPC treatment.

One-pot synthesis of itaconic acid by engineered Halomonas bluephagenesis TDZI-08

Itaconic acid (IA) is one of the twelve high value-added platform compounds applied in various fields including coatings, adhesives, plastics, resins, and biofuels. In this study, we established a one-pot catalytic synthesis system for IA from citric acid based on the engineered salt-tolerant bacterial strain Halomonas bluephagenesis TDZI-08 after investigating factors that hindered the process and optimizing the carbon source, nitrogen source, inducer addition time, and surfactant dosage. The open, non-sterile, one-pot synthesis with TDZI-08 in a 5 L fermenter achieved the highest IA titer of 40.50 g/L, with a catalytic yield of 0.68 g IA/g citric acid during the catalytic stage and a total yield of 0.42 g IA/g (citric acid+gluconic acid). The one-pot synthesis system established in this study is simple and does not need sterilization or aseptic operations. The findings indicate the potential of H. bluephagenesis for industrial production of IA.

Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semipilot scale.

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD+-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.

Genomic Analysis of Aspergillus Section Terrei Reveals a High Potential in Secondary Metabolite Production and Plant Biomass Degradation.

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus so far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.

Balancing pH and yield: exploring itaconic acid production in Ustilago cynodontis from an economic perspective

BackgroundItaconic acid is a promising bio-based building block for the synthesis of polymers, plastics, fibers and other materials. In recent years, Ustilago cynodontis has emerged as an additional itaconate producing non-conventional yeast, mainly due to its high acid tolerance, which significantly reduces saline waste coproduction during fermentation and downstream processing. As a result, this could likely improve the economic viability of the itaconic acid production process with Ustilaginaceae.ResultsIn this study, we characterized a previously engineered itaconate hyper-producing Ustilago cynodontis strain in controlled fed-batch fermentations to determine the minimal and optimal pH for itaconate production. Under optimal fermentation conditions, the hyper-producing strain can achieve the theoretical maximal itaconate yield during the production phase in a fermentation at pH 3.6, but at the expense of considerable base addition. Base consumption is strongly reduced at the pH of 2.8, but at cost of production yield, titer, and rate. A techno-economic analysis based on the entire process demonstrated that savings due to an additional decrease in pH control reagents and saline waste costs cannot compensate the yield loss observed at the highly acidic pH value 2.8.ConclusionsOverall, this work provides novel data regarding the balancing of yield, titer, and rate in the context of pH, thereby contributing to a better understanding of the itaconic acid production process with Ustilago cynodontis, especially from an economic perspective.

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii

BackgroundAmidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO2, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.ResultsIn this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g·L−1 after 5 days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies, we further boosted the itaconic acid production reaching titers of 55 g·L−1 after less than 5 days of methanol feed, while increasing the product yield on methanol from 0.06 g·g−1 to 0.24 g·g−1.ConclusionOur results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

Techno-economics and environmental assessment of sorbitol and itaconic acid production from sugarcane-based feedstock

Production of sorbitol or itaconic acid from sugarcane feedstocks in energy self-sufficient biorefinery scenarios were investigated, via Aspen Plus® simulations, techno-economic and environmental assessments. Sorbitol co-produced with fructose from A-molasses had a minimum selling price (MSP) (0.81 $/kg) similar to technical-grade sorbitol market prices (0.5 to 1.1$/kg), and an internal rate of return (IRR) of 19.65%. Sorbitol co-produced with mannitol from A-molasses had a lower MSP (0.48 $/kg), below food-grade sorbitol prices (0.58$/kg) and a higher IRR (23.63%). Combining A-molasses with lignocelluloses for the co-production of sorbitol and mannitol increased the MSP (0.63 $/kg) above the market price and decreased the IRR (18.46%). This scenario also had greenhouse gas emissions lower than that of its A-molasses only counterpart, mainly due to the method of hydrogen production. Itaconic acid production from sugarcane feedstocks in similar scenarios was unattractive due to lower IRRs (4.49% to 11.61%).

Engineering of itaconic acid pathway via co-localization of CadA and AcnA in recombinant Escherichia coli.

Itaconic acid is an excellent polymeric precursor with a wide range of industrial applications. The efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. However, limitation in the itaconic acid precursor supply was revealed by finding out the key intermediate of the tricarboxylic acid in the itaconic acid pathway. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool to increase itaconic acid productivity are required. In this study, we introduce a synthetic protein scaffold system between CadA and AcnA to physically combine the two enzymes. Through the introduction of a synthetic protein scaffold, 2.1g L-1 of itaconic acid was produced at pH 7 and 37°C. By fermentation, 20.1g L-1 for 48h of itaconic acid was produced with a yield of 0.34gg-1 glycerol. These results suggest that carbon flux was successfully increased itaconic acid productivity.

Quercetin induces itaconic acid-mediated M1/M2 alveolar macrophages polarization in respiratory syncytial virus infection

BackgroundQuercetin has received extensive attention for its therapeutic potential treating respiratory syncytial virus (RSV) infection diseases. Recent studies have highlighted quercetin's ability of suppressing alveolar macrophages (AMs)-derived lung inflammation. However, the anti-inflammatory mechanism of quercetin against RSV infection still remains elusive. PurposeThis study aims to elucidate the mechanism about quercetin anti-inflammatory effect on RSV infection. MethodsBALB/c mice were intranasally infected with RSV and received quercetin (30, 60, 120 mg/kg/d) orally for 3 days. Additionally, an in vitro infection model utilizing mouse alveolar macrophages (MH-S cells) was employed to validate the proposed mechanism. ResultsQuercetin exhibited a downregulatory effect on glycolysis and tricarboxylic acid (TCA) cycle metabolism in RSV-infected AMs. However, it increased itaconic acid production, a metabolite derived from citrate through activating immune responsive gene 1 (IRG1), and further inhibiting succinate dehydrogenase (SDH) activity. While the suppression of SDH activity orchestrated a cascading downregulation of Hif-1α/NLRP3 signaling, ultimately causing AMs polarization from M1 to M2 phenotypes. ConclusionOur study demonstrated quercetin stimulated IRG1-mediated itaconic acid anabolism and further inhibited SDH/Hif-1α/NLRP3 signaling pathway, which led to M1 to M2 polarization of AMs so as to ameliorate RSV-induced lung inflammation.

Improved consolidated bioprocessing for itaconic acid production by simultaneous optimization of cellulase and metabolic pathway of Neurospora crassa

Lignocellulose was directly used in itaconic acid production by a model filamentous fungus Neurospora crassa. The promoters of two clock control genes and cellobiohydrolase 1 gene were selected for heterologous genes expression by evaluating different types of promoters. The effect of overexpression of different cellulase was compared, and it was found that expression of cellobiohydrolase 2 from Trichoderma reesei increased the filter paper activity by 2 times, the cellobiohydrolase activity by 4.5 times, and that the itaconic acid titer was also significantly improved. A bidirectional cis-aconitic acid accumulation strategy was established by constructing the reverse glyoxylate shunt and expressing the transporter MTTA, which increased itaconic acid production to 637.2 mg/L. The simultaneous optimization of cellulase and metabolic pathway was more conducive to the improvement of cellulase activity than that of cellulase alone, so as to further increase itaconic acid production. Finally, through the combination of fermentation by optimized strains and medium optimization, the titers of itaconic acid using Avicel and corn stover as substrate were 1165.1 mg/L and 871.3 mg/L, respectively. The results prove the potential of the consolidated bioprocessing that directly converts lignocellulose to itaconic acid by a model cellulase synthesizing strain.

Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 glucose and 12 acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch.

Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.

Metabolic engineering of Shewanella oneidensis to produce glutamate and itaconic acid

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products.Key points•Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production.•Deletion of ackA/pta redirects carbon flux towards TCA cycle.•Heterologous expression of cadA and acnB enables itaconic acid production.

In situ adsorption of itaconic acid from fermentations of Ustilago cynodontis improves bioprocess efficiency

BackgroundReducing the costs of biorefinery processes is a crucial step in replacing petrochemical products by sustainable, biotechnological alternatives. Substrate costs and downstream processing present large potential for improvement of cost efficiency. The implementation of in situ adsorption as an energy-efficient product recovery method can reduce costs in both areas. While selective product separation is possible at ambient conditions, yield-limiting effects, as for example product inhibition, can be reduced in an integrated process.ResultsAn in situ adsorption process was integrated into the production of itaconic acid with Ustilago cynodontis IAmax, as an example of a promising biorefinery process. A suitable feed strategy was developed to enable efficient production and selective recovery of itaconic acid by maintaining optimal glucose concentrations. Online monitoring via Raman spectroscopy was implemented to enable a first process control and understand the interactions of metabolites with the adsorbent. In the final, integrated bioprocess, yield, titre, and space–time yield of the fermentation process were increased to values of 0.41 gIA/gGlucose, 126.5 gIA/L and 0.52 gIA/L/h. This corresponds to an increase of up to 30% in comparison to the first extended batch experiment without in situ product removal. Itaconic acid was recovered with a purity of at least 95% and high concentrations above 300 g/L in the eluate.ConclusionIntegration of product separation via adsorption into the bioprocess was successfully conducted and improved the efficiency of itaconic acid production. Raman spectroscopy was proven to be a reliable tool for online monitoring of various metabolites and facilitated design and validation of the complex separation and feed process. The general process concept can be transferred to the production of various similar bioproducts, expanding the tool kit for design of innovative biorefinery processes.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high – 120 g/L – glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Utilization of sweet sorghum juice as a carbon source for enhancement of itaconic acid production in engineered Corynebacterium glutamicum

Itaconic acid is a promising biochemical building block that can be used in polymer synthesis. Itaconic acid is currently produced in industry by the natural producer fungus Aspergillus terreus using glucose as a main carbon source. Most research for itaconic acid production using lignocellulosic-based carbon sources was carried out by A. terreus. Engineered Corynebacterium glutamicum strain which can grow in presence of fermentation inhibitors without effect on growth, was used for production of itaconic acid using sweet sorghum juice and bagasse sugar lysate (BSL). BSL contains many inhibitors unlike sorghum juice. C. glutamicum could grow in the media containing both types of lignocellulose-based carbon sources without showing any growth inhibition, however, sorghum juice was better in itaconic acid production than BSL. Different constructed strains of C. glutamicum were used for itaconic acid production, however, C. glutamicum ATCC 13032 pCH-Tad1optAdi1opt strain expressing Adi1/Tad1 genes (trans-pathway) from Ustilago maydis proved to be better in itaconic acid production giving final titer of 8.4 and 4.02 g/L using sweet sorghum juice and BSL as the sole carbon sources by fed-batch fermentation. Our study is the first for production of itaconic acid using sweet sorghum juice and BSL. The present study also proved that C. glutamicum can be used for enhancing itaconic acid production using lignocellulosic-based carbon sources.

Itaconic acid: microbial production using organic wastes as cost-effective substrates

Abstract Itaconic acid is one of industrially important organic acid having wide application in environmental protection, food and textile industries. Microorganisms mainly fungi have vast potential to be exploited for itaconic acid production. But low yield and higher cost of production are major drawback creating a settle back for industrial production. This problem can be solved by using low cost organic waste as substrate. This review summarizes recent research on production of itaconic acid using organic wastes, microorganisms involved, extraction, application and problem faced during utilization of agro-industrial wastes.